This program is designed to help understand the role played by the pterin cofactor, tetrahydrobiopterin, in the mixed function oxygenase reaction of phenylalanine and tyrosine hydroxylase. During the course of these reactions one atom of molecular oxygen is inserted into the aromatic ring of phenylalanine or tyrosine with a synchronous conversion of the tetrahydropterin to an "active" or "dihydro quinonoid" form of the pterin. Pterin reductase, in the presence of NADH, converts the dihydropterin back to the tetrahydro level. With the aid of affinity chromatography, hemogeneous sheep- and rat-liver dihydropterin reductases have been isolated in greater than 30 percent overall yield. Their gross structural features have been examined using polyacrylamide electrophoresis, gel filtration and titration with mercurials. Accurate fluorescent titration studies have suggested the dimeric enzyme has only one site for NADH. This observation contrasts with the results from equilibrium dialysis experiments which suggest the presence of two sites. Binding studies employing radioactively-labeled NADH have been initiated to resolve this problem. In addition fluorescent substrate and pyridine cofactor analogs have been devised which will aid in the characterization of the active sites. BIBLIOGRAPHIC REFERENCE: The Purification of Sheep Liver Dihydropteridine Reductase by Affinity Chromatography. Stephanie Webber and John M. Whiteley, Fed. Proc. 35, 2041 (1976).